The Very Long Chain Fatty Acid (C26:25OH) Linked to the Lipid A Is Important for the Fitness of the Photosynthetic Bradyrhizobium Strain ORS278 and the Establishment of a Successful Symbiosis with Aeschynomene Legumes

Abstract

In rhizobium strains, the lipid A is modified by the addition of a very long-chain fatty acid (VLCFA) shown to play an important role in rigidification of the outer membrane, thereby facilitating their dual life cycle, outside and inside the plant. In Bradyrhizobium strains, the lipid A is more complex with the presence of at least two VLCFAs, one covalently linked to a hopanoid molecule, but the importance of these modifications is not well-understood. In this study, we identified a cluster of VLCFA genes in the photosynthetic Bradyrhizobium strain ORS278, which nodulates Aeschynomene plants in a Nod factor-independent process. We tried to mutate the different genes of the VLCFA gene cluster to prevent the synthesis of the VLCFAs, but only one mutant in the lpxXL gene encoding an acyltransferase was obtained. Structural analysis of the lipid A showed that LpxXL is involved in the transfer of the C26:25OH VLCFA to the lipid A but not in the one of the C30:29OH VLCFA which harbors the hopanoid molecule. Despite maintaining the second VLCFA, the ability of the mutant to cope with various stresses (low pH, high temperature, high osmolarity, and antimicrobial peptides) and to establish an efficient nitrogen-fixing symbiosis was drastically reduced. In parallel, we investigated whether the BRADO0045 gene, which encodes a putative acyltransferase displaying a weak identity with the apo-lipoprotein N-acyltransferase Lnt, could be involved in the transfer of the C30:29OH VLCFA to the lipid A. Although the mutant exhibited phenotypes similar to the lpxXL mutant, no difference in the lipid A structure was observed from that in the wild-type strain, indicating that this gene is not involved in the modification of lipid A. Our results advance our knowledge of the biosynthesis pathway and the role of VLCFAs-modified lipid A in free-living and symbiotic states of Bradyrhizobium strains.

Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency

“…For example, the putative T2SS determined S‐layer could together with the LPS and the hopanoid lipids constitute a multilayered envelope which provides sufficient strength to the bacteria to withstand the NCR peptides or other stresses in the symbiotic nodule cells. Removing the individual components of this envelope could have an imperceptible or moderate effect on the bacteroids (Silipo et al ., ; Kulkarni et al ., ; Busset et al ., ; this work) but, possibly, removing multiple parts simultaneously would be much more deleterious for the bacteroids. Redundancy on the other hand can be achieved by homologous genes or through a functional complementation by an unrelated pathway.…”

“…S9) genes show a shift in expression, either up-or down-regulated, in bacteroids confirming the importance of the cell wall dynamics and the cell cycle regulation during bacteroid differentiation (Supporting Information Appendix S2). Other important features involved in the chronic establishment of the bacteroids are the lipopolysaccharide (LPS) and hopanoid lipids of the membrane since mutants that are affected in the production of these lipids form defective bacteroids (Silipo et al, 2014;Kulkarni et al, 2015;Busset et al, 2016Busset et al, , 2017. However, the genes involved in their biosynthesis show no or only slight differences in expression (Supporting Information Figs S10-S12).…”

Hopanoid lipids: from membranes to plant–bacteria interactions

“…However, it is also possible that the relevance of hopanoids in Bradyrhizobium spp. symbioses can be attributed to the presence of an unusual lipid A structure: hopanoid-lipid A (HoLA), in which lipid A is covalently attached to an extended hopanoid polyol 113,115,116 (FIG. 4).…”

Extended hopanoid loss reduces bacterial motility and surface attachment and leads to heterogeneity in root nodule growth kinetics in a Bradyrhizobium-Aeschynomene symbiosis

“…Other potential extended hopanoid-mediated effects on the symbiosis include regulation of membrane-based processes, such as motility, oxidative phosphorylation, the early legume-rhizobia dialogue or transport across the symbiosome membranes. Specifically, the loss of HoLA from the outer membrane in the B. diazoefficiens ΔhpnH mutant is likely to disrupt processes involving lipid A, which has previously been shown to enhance bacterial fitness in A. afraspera hosts 40 . Identifying the enzyme(s) responsible for the attachment of hopanoids to lipid A will be necessary to determine how much HoLA contributes to the ΔhpnH phenotypes we observed in this work, as well as the universality of these phenotypes to other host-bacteria interactions.…”